CN1108295C - Thiol derivatives with metallopeptidase (ACE/NEP) inhibitory activity - Google Patents

Thiol derivatives with metallopeptidase (ACE/NEP) inhibitory activity Download PDF

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CN1108295C
CN1108295C CN96199355A CN96199355A CN1108295C CN 1108295 C CN1108295 C CN 1108295C CN 96199355 A CN96199355 A CN 96199355A CN 96199355 A CN96199355 A CN 96199355A CN 1108295 C CN1108295 C CN 1108295C
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phenyl
phenyl methyl
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CN1268945A (en
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F·皮拉西尼
S·罗马格纳诺
G·诺西尼
F·萨坦格洛
C·塞米拉洛
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/06—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to the ring carbon atoms
    • C07D333/24—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/54—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/55—Acids; Esters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/22—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C07D277/30—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals

Abstract

Compounds of formula (I) wherein R, R1, R2, R3 have the meanings reported in the description, processes for their preparation and pharmaceutical compositions which contain them as active ingredients are described. The compounds of formula (I) are endowed with a mixed ACE-inhibitory and NEP-inhibitory activity and are useful in the treatment of cardiovascular diseases.

Description

Have metallopeptidase (ACE/NEP) and suppress active mercapto derivatives
The present invention relates to have the mercapto derivatives of metallopeptidase inhibitory activity, more specifically, relate to the N-sulfydryl acyl group alanine derivatives that can be used for the treatment of cardiovascular disorder.
The pharmacology interest that the research metallopeptidase suppresses molecule comes from this enzyme to the horizontal role of the heart recycle system.
In fact people know, have the active compound of angiotensin converting enzyme (ACE) inhibition and mainly can be used for the treatment of hypertension, heart failure and infraction back disease, reason is the formation that they suppress angiotensin II, angiotensin II is a kind of material with several effects, comprising elevation of blood pressure.
Have the active compound of endothelin-converting enzyme (ECE) inhibition and can be used as the anti-vasoconstriction medicine, reason is that their suppress the formation of endothelium peptide, and the endothelium peptide is a kind of 21 amino acid whose peptides with vasoconstrictor activity.
Can be used as vasodilator and have the active compound of inhibition that neutral endopeptidase (NEP) also is referred to as enkephalinase, reason is that the NEP enzyme not only makes endogenous brain deltorphin delta inactivation but also makes some natriuretic factor inactivations, a kind of hormone of atrial natriuretic peptide wherein (ANF) for example by heart secretion, it can strengthen vasorelaxation, and increases diuresis and natriuresis drainage on the kidney level.
Therefore, even they act on cardiovascular systems with the different mechanism of action, usually also be these compounds with metallopeptidase inhibitory activity to be used for separately or in conjunction with being used for the treatment of hypertension, renal failure, congestive heart failure and ischemic heart disease.
In the mercapto derivatives inhibitor of metallopeptidase, by people such as Roques at Nature (nature), Vol.288, the 286-288 page or leaf, (1980) Thiorphan ((DL-(3-sulfydryl-2-benzyl propionyl) glycine) and captopril (Captopril) (the The Merck Index that describe for the first time in, XI version, No.1773, the 267th page) to be considered to be respectively the parent compound of nep inhibitor and ACE inhibitor.
Other molecule that contains sulfhydryl structure with metallopeptidase inhibitory activity has been described in the document.
People such as S.R.Bertenshaw are at Bioorganic ﹠amp; Medicinal ChemistryLetters (biological organic and medical chemistry communication), 10,1953-1958 has described in 1993 and has ECE and suppress active N-sulfydryl acyl group dipeptides, particularly is the N-sulfydryl acyl derivative of tryptophane.
US patent No.4,401,677 (E.R.Squibb ﹠amp; Sons Inc.) has described and has enkephalinase and suppress the acid of active sulfydryl alkanoyl amido.
US patent No.4,199,512 (E.R.Squibb ﹠amp; Sons.Inc.) described and have ACE and suppress the acid of active sulfydryl alkanoyl amido.
European patent application No.0566157 (Schering Corporation) has described has the N-sulfydryl acyl derivative that NEP suppresses active L-Ala.
European patent application No.0449523 (E.R.Squibb ﹠amp; Sons Inc.) has described and has NEP and suppress active sulfydryl or acyl sulfenyl fluoroform acid amides.
European patent application No.0524553[Institut National de la Sante et de laRecherche Medicale (INSERM)] described and had neutral endopeptidase and peptidyl peptidase A suppresses active acyl group sulfydryl alkane acyl dipeptides.
International Patent Application WO 93/08162[Rhone-Poulenc Rorer S.A.-InstitutNational de la Sante et de la Recherche Medicale (INSERM)] described and have ACE/NEP and mix to suppress active β, β-disubstituted alpha-mercapto methyl propanamide.
European patent application No.0419327 (Societe Civile Bioproject) has described has enkephalinase and ACE suppresses active amino acid derivative, for example the N-sulfydryl acyl derivative of phenylalanine, Histidine and tryptophane.
People such as S.S Bhagwat are at Bioorganic ﹠amp; Medicinal Chemistry Letters (biological organic and medical chemistry communication), 7,735-738 has also described in 1995 and has ACE-and suppress active and NEP-suppresses active alpha-mercapto acyl group dipeptides.
In the end in this piece document; the author reaches a conclusion; mix and suppress active although the existence of xenyl methyl can make the molecule that contains alpha-mercapto acyl group dipeptides structure have required ACE/NEP, use as α-or this class group substituted biphenyl base of betanaphthyl can cause active remarkable forfeiture.
Now, we find N-sulfydryl acyl group alanine derivatives that angiotensin converting enzyme and neutral endopeptidase are had remarkable inhibiting activity (ACE/NEP dual restraining activities) to make them can be used in particular in the treatment of cardiovascular disorder.
Therefore, the objective of the invention is formula I compound and pharmacologically acceptable salt thereof,
Wherein
R is sulfydryl or the R that can be converted into sulfydryl in vivo 4The COS group;
R 1Be straight or branched C 2-C 4Alkyl or aryl or moieties have the aralkyl of 1 to 6 carbon atom; wherein aryl is phenyl or has 1 or 2 the heteroatomic 5 or 6 Yuans fragrant heterocycles that are selected from N, O and S; randomly replaced by one or more substituting groups; these substituting groups are identical or different, are selected from halogen atom, hydroxyl, alkoxyl group, alkyl, alkylthio, moieties the alkyl sulphonyl or the alkoxy carbonyl of 1 to 6 carbon atom arranged, contain the C of one or more fluorine atoms 1-C 3Alkyl, carboxyl, nitro, amino or aminocarboxyl, amido, amino-sulfonyl, moieties have list or dialkyl amido or the list or the dialkyl amino carbonyl of 1 to 6 carbon atom;
R 2Be hydrogen atom, straight or branched C 1-C 4Alkyl or benzyl;
R 3Be to have 1 or 2 heteroatomic 5 Yuans or 6 Yuans fragrant heterocycles that are selected from N, O and S, randomly replaced by phenyl, this phenyl and heterocyclic radical are randomly replaced by one or more identical or different substituting groups, and described substituting group is selected from halogen atom, alkyl, alkoxyl group, moieties 1 alkylthio or alkoxy carbonyl to 3 carbon atoms;
R 4Be straight or branched C 1-C 4Alkyl or phenyl;
The carbon atom that indicates asterisk is a stereogenic centres;
Condition is R 3Not imidazolyl or indyl.
Formula I compound contains two stereogenic centres, can exist with the form of steric isomer thus.
Therefore, the formula I compound that the objective of the invention is to be the three-dimensional heterogeneous mixture form and be the single stereoisomers form.
The formula I compound of the object of the invention has the ACE/NEP dual restraining activities, can be used for the treatment of cardiovascular disorder.
In this specification sheets, except as otherwise noted, term straight or branched alkyl refers to such as this class alkyl of methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, sec-butyl, the tertiary butyl, isobutyl-, n-pentyl, 2-amyl group, 3-amyl group, isopentyl, tert-pentyl, n-hexyl and isohexyl; Term straight or branched alkoxyl group refers to such as these alkoxyl groups of methoxyl group, oxyethyl group, positive propoxy and isopropoxy; The term halogen atom refers to fluorine, chlorine, bromine or iodine atom; The term acyl group refers to by aliphatic series or aromatic carboxylic acid such as acetate, propionic acid, butyric acid and phenylformic acid deutero-acyl group; Term has 1 or 2 heteroatomic 5 Yuans or 6 Yuans fragrant heterocycles that are selected from N, O and S and refers to randomly condense with benzene such as thiazole, isoxazole, oxazole, isothiazole, pyrazoles, imidazoles, thiophene, pyrroles, pyridine, pyrimidine, pyrazine, pyridazine and furans.
The example of formula I compound pharmacologically acceptable salt has salt that forms with basic metal or alkaline-earth metal and the salt that forms with pharmaceutically acceptable organic bases.
Preferred formula I compound is R wherein 3Be to have 1 or 2 compound that is selected from heteroatomic 5 Yuans or the 6 Yuans aromatic heterocyclics of N, O and S.
More preferred this compounds is R wherein 1Be the formula I compound of phenylalkyl, wherein phenylalkyl is randomly replaced by one or more identical or different substituting group that is selected from halogen atom, hydroxyl, alkyl or alkoxyl group.
The example of preferred formula I compound pharmacologically acceptable salt is the salt that forms with basic metal such as sodium, lithium and potassium.
It will be readily apparent to one skilled in the art that wherein R is the R that can be converted into sulfydryl in organism 4The formula I compound of COS group and R wherein 2For the formula I compound of alkyl or benzyl is sulfydryl (R=SH) or R for R wherein 2Be hydrogen atom (R 2=H) the bioprecursor (prodrug) of corresponding formula I compound.
The example of the concrete preferred formula I compound of the present invention is:
N-(3-phenylcarbonyl group sulfenyl-2-phenyl methyl propionyl)-(2-thienyl)-alanine methyl ester;
N-(3-phenylcarbonyl group sulfenyl-2-phenyl methyl propionyl)-(4-thiazolyl)-alanine methyl ester;
N-(3-phenylcarbonyl group sulfenyl-2-phenyl methyl propionyl)-(2-pyridyl)-alanine methyl ester;
N-(3-phenylcarbonyl group sulfenyl-2-phenyl methyl propionyl)-(3-pyridyl)-alanine methyl ester;
N-(3-phenylcarbonyl group sulfenyl-2-phenyl methyl propionyl)-(2-furyl)-alanine methyl ester;
N-(2-phenyl methyl-3-mercapto radical propionyl group)-(2-thienyl)-L-Ala;
N-(2-phenyl methyl-3-mercapto radical propionyl group)-(4-thiazolyl)-L-Ala;
N-(2-phenyl methyl-3-mercapto radical propionyl group)-(2-pyridyl)-L-Ala;
N-(2-phenyl methyl-3-mercapto radical propionyl group)-(3-pyridyl)-L-Ala;
N-(2-phenyl methyl-3-mercapto radical propionyl group)-(2-furyl)-L-Ala.
The preparation of formula I compound of the present invention is undertaken by synthesis method, and this method comprises the reaction between the alanine derivatives of formula II compound and formula III,
Figure C9619935500081
Wherein R and R 1Implication is the same;
Figure C9619935500082
R wherein 2And R 3Implication is the same.
Carry out condensation reaction according to the chemistry of peptides ordinary method.
Before reacting, it is useful will disturbing the selectivity functional group of reacting to carry out due care.
Carry out selective protection according to conventional methods.
In this respect, preferably inciting somebody to action wherein, R is R 4The compound of COS group obtains wherein R=R thus as the intermediate of formula II 4The corresponding formula I compound of COS can obtain the wherein formula I compound of R=SH by it by hydrolysis.
According to the reaction of being carried out and the functional group that will protect, all belong to knowledge well known to those skilled in the art to the assessment of the profitability of selective protection effect with to the selection of the protection kind adopted.
Carry out removing of selective protection base according to conventional methods.
At the summary reference of the use of protecting group in the organic chemistry " Protective Groups in OrganicSynthesis (protecting group in the organic synthesis) ", John Wiley ﹠amp referring to Theodora W.Greene and Peter G.M.Wuts; Sons, Inc., II version, 1991.
Formula II and formula III compound are known, or can prepare easily according to conventional methods.
For example, can be according to E.R.Squibb ﹠amp; The described method preparation formula of the English Patent NO.1576161 of Sons Inc. II compound.
Prepare the formula I compound that is the single stereoisomers form by stereospecific synthesis or by separating three-dimensional heterogeneous mixture according to conventional methods.
A further object of the invention is the salt according to the ordinary method preparation I compound.
Formula I compound has the ACE/NEP dual restraining activities as purpose of the present invention, can be used for the treatment of cardiovascular disorder.
Inhibition activity by in vitro tests assessment formula I compound.
Especially formula I compound and aforesaid Thiorphan and captopril are compared the inhibition activity of assessing formula I compound.
With IC 50The vitro inhibition activity of the formula I compound of value representation is the pharmacology significance, because it is nM concentration.
Described activity can be compared with captopril with regard to its ACE suppresses activity, can compare with Thiorphan with regard to its NEP suppresses activity.
When being applied in the treatment, formula I compound can be mixed with solid or the composition of liquid medicine that is suitable for oral or administered parenterally.
Therefore, containing the formula I compound for the treatment of significant quantity and a kind of pharmaceutical composition of pharmaceutical carrier is another object of the present invention.
According to the present invention, the object lesson of pharmaceutical composition is to be suitable for oral tablet, coated tablet, capsule, granule, solution and suspension, is suitable for the solution and the suspension of administered parenterally.
The pharmaceutical composition for preparing the object of the invention according to conventional methods.
The per daily dose of formula I compound or corresponding prodrug depends on Several Factors such as severity of disease, patient's individual reaction or type of preparation, but is generally per kilogram of body weight 0.1mg-10mg, is divided into single dose or a plurality of per daily dose.
The present invention now provides following examples for explanation.
Except as otherwise noted, use the flash chromatography silica gel of Baker company (numbering 7024-00) to carry out flash chromatography.
Embodiment 1
N-(3-phenylcarbonyl group sulfenyl-2-phenyl methyl propionyl)-(4-thiazolyl) The preparation of-L-alanine methyl ester (compound 1)
Under 0 ℃ of agitation condition to 3-phenylcarbonyl group sulfenyl-2-phenyl methyl propionic acid (2.78g; 9.25mmol), (4-thiazolyl)-L-alanine methyl ester (2.40g; 9.25mmol), triethylamine (2.58ml; 18.5mmol) and the mixture that forms of tetrahydrofuran (THF) (25ml) and methylene dichloride (40ml) in add hydroxybenzotriazole (1.25g; 9.25mmol) tetrahydrofuran (THF) (30ml) solution, add dicyclohexylcarbodiimide (1.81g subsequently; 9.25mmol) methylene dichloride (10ml) solution, here (4-thiazolyl)-L-alanine methyl ester is by handling N-tertbutyloxycarbonyl-(4-thiazolyl)-L-alanine methyl ester (by Sinthetech Inc., Oregon provides) with methyl alcohol and thionyl chloride and obtaining.
Under the stirring at room condition, reaction mixture was kept 20 hours, leach dicyclohexylurea (DCU) then, and solvent evaporated under reduced pressure.Collect resistates with ethyl acetate, solution is with 20% sodium-chlor, 5% sodium bicarbonate and wash with 20% sodium chloride aqueous solution once more.
After being separated and evaporating organic phase, by flash chromatography (silica gel, eluent ethylacetate: hexane=1: 1, nitrogen is pressed 0.1 normal atmosphere) purifying gained solid, acquisition is N-(3-phenylcarbonyl group sulfenyl-2-phenyl methyl propionyl)-(4-thiazolyl)-L-alanine methyl ester (3.85g of oily matter thus; Yield 88%).
Handle this oily matter with ether and hexane, subsequent filtration obtains solid state purpose product (2.2g; Stereoisomerism is than 2S: 2R=85: 15).
1H-NMR (200MHz, CDCl 3): δ (ppm): 2.67-3.30 (m, 7H, CH 2-CH-CH 2, C H 2-thiazolyl); 3.60 (s, 3H, OCH 3); 4.77-4.89 (m, 1H, CONH-CH); 6.53 (s, 1H, NH); (7.15-7.90 m, 10H, phenyl); (6.92-8.51 m, 2H, thiazolyl).
From mother liquor, isolate the compound 1 (1.5g of another part; Three-dimensional foreign matter is than 2S: 2R=65: 35), be oily matter.
Prepare following compound with similar method:
N-(3-phenylcarbonyl group sulfenyl-2-phenyl methyl propionyl)-(2-thienyl) -L-alanine methyl ester (compound 2)
Fusing point 68-70 ℃
1H-NMR (200MHz, CDCl 3): δ (ppm): 2.65-3.40 (m, 7H, CH 2-CH-CH 2, C H 2-thienyl); 3.61 and 3.62 (2s, 3H, OCH 3); 4.70-4.90 (2m, 1H, CONH-CH); 5.85-5.90 and 5.95-6.02 (2m, 1H, NH); (6.60-7.05 m, 3H, thienyl); (7.10-8.00 m, 10H, phenyl);
N-(3-phenylcarbonyl group sulfenyl-2-phenyl methyl propionyl)-(2-pyrrole The pyridine base)-L-alanine methyl ester (compound 3)
Fusing point 84-86 ℃
1H-NMR (200MHz, CDCl 3): δ (ppm): 2.70-3.30 (m, 7H, CH 2-CH-CH 2, C H 2-pyridyl); 3.59 (s, 3H, OCH 3); 4.71-4.92 (m, 1H, CONH-CH); (6.90-8.28 m, 15H, NH, phenyl, the fixed base of pyrrole).
Embodiment 2
N-(2-phenyl methyl-3-mercapto radical propionyl group)-(4-thiazolyl)- The preparation of L-L-Ala (compound 4)
Will be according to N-(3-phenylcarbonyl group sulfenyl-2-phenyl methyl propionyl)-(4-thiazolyl)-L-alanine methyl ester (1.20g of embodiment 1 described preparation; 2.56mmol) be suspended in the ethanol (30ml), by being blown into the nitrogen exhaust to remove oxygen.
Under 0 ℃ of condition of nitrogen gas, in this suspension, drip the aqueous sodium hydroxide solution 1N (7.68ml) that took off gas.
Under the agitation condition reaction mixture was remained in room temperature 4 hours, then 0 ℃ of cooling down, with 10% hydrochloric acid (5ml) of the degassing in advance and the solution acidifying of water (5ml).
The reduction vaporization mixture is to dry, and drying is collected and be evaporated to once more to resistates with acetonitrile.
Use methyl alcohol: methylene dichloride=1: 1 mixture (15ml) collection resistates, this resistates is filtered to remove inorganic salt, reduction vaporization solution is to dry.
Use the water of hydrochloric (20ml) and methylene dichloride (20ml) to collect resistates then.
Separate two-phase, the organic phase reduction vaporization obtains resistates (0.81g) to dry; Handle, filter and drying with uncle's butyl ether, obtain N-(2-phenyl methyl-3-mercapto radical propionyl group)-(4-thiazolyl)-L-L-Ala (0.52g; Yield 58%; Stereoisomerism is than 2S: 2R=90: 10).
Fusing point 145-147 ℃
1H-NMR (200MHz, CDCl 3): δ (ppm): 1.53 (t, 1H, JHH=8.4Hz, SH); 2.51-2.98 (m, 5H, CH 2-CH-CH 2); 3.39-3.51 (ABX, 2H, Jab=14.7Hz, Jax=3.4Hz, Jbx=5.9Hz, C H 2-thiazolyl); 4.63-4.71 (m, 1H, CHCOO); 6.51 (d, 1H, JHH=5.8Hz, CONH); 7.09-7.30[m, 6H, phenyl, CH (thiazolyl)]; 8.85[d, 1H, JHH=2.0Hz, CH (thiazolyl)].
Compound below the similar approach preparation:
N-(2-phenyl methyl-3-mercapto radical propionyl group)-(2-thienyl)- L-L-Ala (compound 5)
1H-NMR (200MHz, CDCl 3): δ (ppm): 1.40-1.61 (m, 1H, SH); 2.47-3.46 (m, 7H, CH 2-CH-CH 2, CH 2-CH-COO); 4.78-4.91 (m, 1H, CHCOO); 6.00-6.06 (m, 1H, CONH); (6.40-7.33 m, 8H, aromatic series); 7.50 (bs, 1H, COOH);
N-(2-phenyl methyl-3-mercapto radical propionyl group)-(2-pyridyl)- L-L-Ala (compound 6)
Fusing point 164-166 ℃
1H-NMR (200MHz, DMSO-d 6): δ (ppm): 1.78-1.86 (m, 1H, SH); 2.20-2.89 (m, 5H, CH 2-CH-CH 2); 2.95-3.26 (m, 2H, C H 2-pyridyl); 4.64-4.75 (m, 1H, CHCOO); 8.33 (d, 1H, JHH=8.1Hz, NH); (7.10-8.47 m, 9H, aromatics); 12.7 (bs, 1H, COOH).
Embodiment 3
The assessment of external pharmacologically active
A) It is active that NEP-suppresses
Be evaluated at according to people such as T.Maeda at Biochim, Biophys.Acta 1983 (biological chemistry biophysical journal), 731 (1), the NEP-in the kidney of rats cortex film of the method preparation described in the 115-120 suppresses active.
Excise kidney from the male Sprague-Dawley rat of heavily about 300g, and be stored in 4 ℃.
Carefully peel renal cortex, fine chopping also is suspended in the damping fluid that homogenizes with the ratio of 1: 15 weight/volume and (contains 1mM MgCl 2, 30mM NaCl, 0.02%NaN 3The 10mM sodium phosphate of pH7.4).
Use the Ultra-Turrax homogenizer will organize cold homogenize 30 seconds then.
The tissue homogenate of about 10ml is at 10ml sucrose (41% weight/volume) higher slice, and in the rotor of a fixed angle under 4 ℃ with 31200rpm centrifugal 30 minutes.
From damping fluid/sucrose interface collection membrane, with 50mM TRIS/HCl damping fluid (pH7.4) washed twice, resuspending is used for-80 ℃ and preserves aliquots containig down in same buffer, until use.
According to people such as C.Llorens at Eur.J.Pharmacol. (European pharmacology magazine), 69, (1981), method described in the 113-116 is assessed NEP and is suppressed active, as described below.
Film suspension aliquots containig (the protein concentration 5 μ g/ml) preincubation that in the presence of 30 ℃ of following amastatins (Bestatin-1mM), will divide by above-mentioned preparation 10 minutes.
Add ( 3H) (Leu 5)-enkephalin (15nM) and damping fluid TRIS/HClpH7.4 (5mM) are to obtain the final volume of 100 μ l.
After 20 minutes, stop enzyme reaction at 30 ℃ of incubations by adding HCl 0.1M (100 μ l).
After polystyrene columns (Porapak Q) goes up the unreacted substrate of chromatographic separation, by liquid scintillation measure relative radioactivity come quantitative meta-bolites ( 3H) formation of Tyr-Gly-Gly.
With respect to untreated membrane product, the inhibition percentage table that meta-bolites in the membrane product of the formula of use I compound and the processing of use control compounds is formed is shown IC 50(nM) value.
B) ACE-suppresses active
According to people such as B.Holmquist at Analytical Biochemistry 95It is active that institute's reported method is assessed the ACE inhibition in (analytical biochemistry), 540-548 (1979).
In thermostatic bath under 37 ℃ with 50 μ M ACE (purifying from rabbit lung (lung rabbit), 250mU/ml, EC 3.4,15.1 SIGMA) with 50 μ l formula I compounds or 50 μ l control compounds or related vector preincubation.
By adding 500 μ l furyl acryloyl phenyl alanyl glycylglycine 0.8mM (FAPGG-SIGMA) initial actions.
Simultaneously, by the Beckman DU-50 spectrophotometer that uses to provide the program of Δ A/ minute of being used to calculate the enzyme kinetics curve and regression coefficient, the absorbancy under the continuous recording 340nm 5 minutes.
With respect to goods, be represented as IC with formula I compound or with inhibition percentage ratio in the goods of control compounds incubation with the carrier incubation 50(nM) value.
Below we have reported allied compound 4,5 and 6 and the ACE of control compounds Thiorphan and captopril suppresses activity and NEP suppresses active IC in the table 1 50(nM) value.
Table 1
The ACE of compound 4, compound 5, compound 6, Thiorphan and captopril suppresses activity and NEP suppresses active, with IC 50(nM) expression
Compound ACE-suppresses active IC 50(nM) NEP-suppresses active IC 50(nM)
4 12 4.7
5 6.5 5.5
6 23 7.1
Thiorphan 99 18
Captopril 4.6 Non-activity
The data that table 1 is reported show, have significant ACE/NEP dual restraining activities as the Compound I of the object of the invention.Described activity can be compared with captopril with regard to its ACE suppresses activity, can compare with Thiorphan with regard to its NEP suppresses activity.

Claims (12)

1. formula I compound and pharmacologically acceptable salt thereof,
Figure C9619935500021
Wherein
R is sulfydryl or the R that can be converted into sulfydryl in vivo 4The COS group;
R 1Be the aralkyl that moieties has 1 to 6 carbon atom, wherein aryl is a phenyl;
R 2Be hydrogen atom, straight or branched C 1-C 4Alkyl;
R 3Be to have 1 or 2 heteroatomic 5 Yuans or 6 Yuans fragrant heterocycles that are selected from N, O and S,
R 4Be straight or branched C 1-C 4Alkyl or phenyl;
The carbon atom that indicates asterisk is a stereogenic centres;
Condition is R 3Not imidazolyl or indyl.
2. the formula I compound of the claim 1 of the salt form that forms with the basic metal that is selected from sodium, lithium and potassium.
3. method for preparing the formula I compound of claim 1 comprises the reaction between formula II compound and formula III alanine derivatives:
Figure C9619935500022
Wherein
R and R 1Implication is described with claim 1;
Wherein
R 2And R 3Implication is described with claim 1.
4. pharmaceutical composition comprises formula I compound and a kind of pharmaceutically acceptable carrier of the claim 1 for the treatment of significant quantity.
5. the pharmaceutical composition that is used for the treatment of the claim 6 of cardiovascular disorder.
6. the application of the formula I compound of claim 1 in the medicine of preparation treatment cardiovascular disorder.
7. according to the formula I compound of claim 1, it is N-(3-phenylcarbonyl group sulfenyl-2-phenyl methyl propionyl)-(4-thiazolyl)-L-alanine methyl ester.
8. according to the formula I compound of claim 1, it is N-(3-phenylcarbonyl group sulfenyl-2-phenyl methyl propionyl)-(2-thienyl)-L-alanine methyl ester.
9. according to the formula I compound of claim 1, it is N-(3-phenylcarbonyl group sulfenyl-2-phenyl methyl propionyl)-(2-pyridyl)-L-alanine methyl ester.
10. according to the formula I compound of claim 1, it is N-(2-phenyl methyl-3-mercapto radical propionyl group)-(4-thiazolyl)-L-L-Ala.
11. according to the formula I compound of claim 1, it is N-(2-phenyl methyl-3-mercapto radical propionyl group)-(2-thienyl)-L-L-Ala.
12. according to the formula I compound of claim 1, it is N-(2-phenyl methyl-3-mercapto radical propionyl group)-(2-pyridyl)-L-L-Ala.
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